CN110423773A - A kind of hydrogen peroxide whole-cell biological sensor and its preparation and application - Google Patents
A kind of hydrogen peroxide whole-cell biological sensor and its preparation and application Download PDFInfo
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Abstract
The present invention relates to a kind of hydrogen peroxide whole-cell biological sensor and its preparation and application.The present invention constructs pCM-lux carrier first, then constructs pCM-lux-PahpC plasmid, then the pCM-lux-PahpC plasmid built is transferred in pseudomonas putida Pseudomonas putida KT2440, obtains hydrogen peroxide whole-cell biological sensor KT2440OX.The bacterium solution of KT2440OX bacterial strain is added in hydrogen peroxide sample to be measured, the relative luminous intensity of sample is read, utilizes the H of known concentration2O2Relative luminous intensity after the bacterium solution of KT2440OX bacterial strain is added in sample, establishes H2O2Relationship between concentration and relative luminous intensity recycles the relative luminous intensity of sample to calculate the H in sample2O2Concentration.Compared with prior art, the present invention can establish science, accurate, quickly detection content of hydrogen peroxide method, especially suitable for remaining hydrogen peroxide in direct test sample, corresponding examination criteria is formulated for national correlation department, some opinions and help are provided, are the detection methods of a very promising residual quantities of hydrogen peroxide.
Description
Technical field
The invention belongs to technical field of bioengineering, more particularly, to a kind of hydrogen peroxide whole-cell biological sensor and its
Preparation and application.
Background technique
Hydrogen peroxide (Hydrogen peroxide), aqueous solution are also referred to as hydrogen peroxide, molecular formula H2O2, sterling is
Colourless liquid, density 1.442g/cm3, -0.41 DEG C of fusing point, 150.2 DEG C of boiling point.There is explosive and corrosivity, while there is oxidation
Property and reproducibility, sterling it is relatively stable, be a kind of strong oxidizer, when contain trace impurity when, illumination can be catalyzed its decomposition, be in
Faintly acid, oxidizing and reproducibility (Tanner PA, Wong AYS.1998.Spectrophotometric
determination of hydrogen peroxide in rainwater.Analytica Chimica Acta 370:
279-287.).In people's daily life everywhere it can be seen that the figure of hydrogen peroxide, it is a kind of oxygen of function admirable
Agent, bleaching agent, disinfectant and crosslinking agent.Hydrogen peroxide is that a kind of very important industrial chemicals is answered since nineteen ninety
Be gradually expanded from initial textile industry to many industries with range, such as chemical industry, electronic product, textile, food (Chen Dihang,
The application field and main production process Chemical Engineering Technology and exploitation 40:32-35. of Li Mianqing .2011. hydrogen peroxide).
Hydrogen peroxide all attaches great importance to its R&D and production as a kind of highly important industrial chemicals, countries in the world, and 90
After age, application range is gradually expanded from initial textile printing and dyeing.The efficient sterilizing of hydrogen peroxide, oxidation bleaching effect and easily
The characteristics of decomposition, low-residual, promotes many food enterprises excess or illegally adds hydrogen peroxide, for killing food, packaging
The happy sense of color of microorganism and increase food in material and equipment container.Hydrogen peroxide due to efficient sterilizing, low-residual, easily
It decomposes, the feature that bleaching effect is good, promotes numerous food product manufacturer and industrial enterprise in food production, processing, packaging material
Using hydrogen peroxide, for killing the harmful microorganism in production equipment, packaging material and food.However, peroxide is excessively used
The phenomenon that changing hydrogen and illegal addition industrial hydrogen peroxide is very serious, frequent occurrence so as to cause food poisoning.As recently
News report in individual illegal retailers chicken feet, brewed sharkskin, brewed toxic is processed with non-food stuff additive hydrogen peroxide
Pig outer skin, bleaching American pistachios, bubble crab meat grain etc. similar food safety affair.Due to the property of hydrogen peroxide itself and other
The influence of factor, hydrogen peroxide is extremely unstable, and the residual quantity after food processing in food is very low, existing hydrogen peroxide inspection
Survey method there is sensitivity low, poor accuracy is unsuitable for the problems such as trace detection.Therefore it establishes a kind of efficient, sensitive, quick
The detection technique for detecting hydrogen peroxide in food, is the key technical problem in the urgent need to address of food safety, to peroxidating
The study on determination method of hydrogen and application, have great importance (the detection method research of hydrogen peroxide in Sun Jia .2014. food
Jilin Agriculture University).
Show in the food quality inspection report carried out at home in part beans fabricated product, Flour product, fish ball, salt
There is the hydrogen peroxide (hydrogen peroxide) of excess to remain in teal, Boiled chicken with salt etc.；Height has been also detected that in the shark's fin of the Hong Kong Special Administrative Region
Up to 1.5% hydrogen peroxide.In China, food hygiene law regulation: there must not be residual peroxide in food and health care product.On ground
It is fresh-keeping that 6mg/L hydrogen peroxide below can be added in Inner Mongolia Autonomous Region and Border in Harbin Area in raw milk by, which only allowing on domain,
Agent, and country is without allowing to make in the food such as Omasum, tripe, jellyfish, sea cucumber, fish-skin, duck's foot, the squid in these areas
With.With social progress and improvement of living standard, the residual harm about hydrogen peroxide is increasingly attracted much attention, state
Inside and outside many studies have shown that side effect is very big after over hydrogenation hydrogen enters human body, its direct stimulation mucosal tissue can also be a wide range of
Ground damages human body cell, accelerates the aging of human body, causes (Zhou Jianhong, Li Hui, Song Shucai, Chen Jinyan, Du Shaohui, Lee such as cancer
Yi Wei, Chen Dongfeng .2005. the hydrogen peroxide Guangdong influence medicine active on Proliferation of Bone Mesenchymal Stem Cells: 1199-
1200.)。
In environmental area, after hydrogen peroxide is discharged into water body and soil, concentration is lower.In the food industry, due to
After hydrogen peroxide passes through simple process, residual quantity is also general lower, the measurement method of China's existing residual peroxide at present
The requirement of the food-safe detection of people is no longer satisfied with detection sanitary standard.Therefore in food hydrogen peroxide it is quick,
Sensitive, quantitative detection is environment protection field and field of food safety key technology difficulty in the urgent need to address.
Present analysis H2O2Method can substantially be divided into chemical analysis method and instrument analytical method.
For the sample of high concentration, chemical titration analysis method is generally used, mainly there is permanganimetric method, iodimetric titration and sulfuric acid
Cerium method etc. (the research Chemical Engineer of Wang Ruibin .2005. accurate determination of hydrogen peroxide content: 62-64.).
Instrument analytical method has high performance liquid chromatography, ultraviolet spectrophotometry, flow injection-spectrophotometry etc..Instrument
Device analysis method belongs to Analytical Methods of Trace, suitable for analyze low concentration sample (Liu Haifeng .2009. quickly detects mistake in food
The remaining bio-sensing method of hydrogen oxide establishes Shanghai Communications University).
Summary of the invention
The object of the invention is in order to provide a kind of full cell of the hydrogen peroxide based on pseudomonad and lux bioluminescence gene
Biosensor and its preparation and application.
Through the invention, science, accurate, quickly detection content of hydrogen peroxide method can be established, especially suitable for direct
Remaining hydrogen peroxide in test sample formulates corresponding examination criteria for national correlation department, provides some opinions and help,
It is the detection method of a very promising residual quantities of hydrogen peroxide.
The purpose of the present invention can be achieved through the following technical solutions:
The present invention provides a kind of preparation method of hydrogen peroxide whole-cell biological sensor, comprising the following steps:
(1) pCM-lux carrier is constructed:
The connection of lux reporter gene is entered into pCMgfp-lacZ plasmid using the method for homologous recombination, building pCM-lux is carried
Body；Wherein, pCMgfp-lacZ is known plasmid, in document Zhang WM, Zhang JJ, Jiang X, Chao HJ, Zhou
NY.2015.Transcriptional activation of multiple operons involved in para-
nitrophenol degradation by Pseudomonas sp strain WBC-3.Appl Environ Microbiol
It is once had been reported that in 81:220-230.；
Expanding primer used by lux reporter gene is
Lux gene forward direction amplimer:
cagaaagcatgccggggatccCTCGAGCTGCAGACTAGTAGGCTTGGAGGATACGTA
TGAC
The reversed amplimer of lux gene:
cgtaatcatggtcatggatcTCAACTATCAAACGCTTCGGTTAA
Wherein underscore lowercase sequence is the homology arm sequence on carrier, and tilted capital letter is to be added by primer
Multiple cloning sites sequence；
(2) pCM-lux-PahpC plasmid is constructed:
Using the method for homologous recombination by ahpC gene in pseudomonas putida Pseudomonas putida KT2440
Promoter PahpC connection enters pCM-lux plasmid, constructs pCM-lux-PahpC plasmid；
Wherein, pseudomonas putida Pseudomonas putida KT2440 is known bacterial strain, in document Song Y,
Hahn T,Thompson IP,Mason TJ,Preston GM,Li G,Paniwnyk L,Huang
WE.2007.Ultrasound-mediated DNA transfer for bacteria.Nucleic Acids Res 35:
E129. there is disclosure in, the promoter PahpC sequence of ahpC gene in pseudomonas putida Pseudomonas putida KT2440
Known to column.
The primer used when expanding promoter are as follows:
The positive amplimer of PahpC promoter
ahpC ins cagaaagcatgccggggatccGAGCCCCTCCTTGAATGTCG
The reversed amplimer of PahpC promoter
ahpC ina agtctgcagctcgagggatccTCATGGGGTTGGAATCAGTTG。
(3) by after the pCM-lux-PahpC carrier built sequencing confirmation, it is false single that stench is transferred to using Ultrasonic transformation method
In born of the same parents bacterium Pseudomonas putida KT2440, the bacterial strain containing pCM-lux-PahpC carrier is referred to as KT2440OX bacterium
Strain, as hydrogen peroxide whole-cell biological sensor.
The pCM-lux carrier is used for through chemiluminescence detection promoter activity, which overcome document Zhang WM,
Zhang JJ,Jiang X,Chao HJ,Zhou NY.2015.Transcriptional activation of multiple
operons involved in para-nitrophenol degradation by Pseudomonas sp strain
PCMgfp-lacZ carrier can only utilize lacZ galactoside in WBC-3.Appl Environ Microbiol 81:220-230.
Enzymatic activity detects the limitation of substrate to be measured as reporting system, realizes the big Reporter System of lux-lacZ-xylE tri-
It connects (wherein, the two genes of lacZ-xylE are already existing in pCMgfp-lacZ plasmid, and lux connection is entered),
Great convenience is provided for follow-up signal detection.
In addition, being easy to appear and being difficult to for there was only the two restriction enzyme sites of BamHI and SphI on pCMgfp-lacZ carrier
The situation of restriction enzyme site is chosen, the new support of building increases the multiple cloning sites of BamHI, XhoI, PstI, SpeI, is subsequent
Clone provides convenience.
The present invention obtains the bacterial strain containing pCM-lux-PahpC carrier according to above-mentioned preparation method and is referred to as KT2440OX
Bacterial strain, as hydrogen peroxide whole-cell biological sensor.
The present invention also provides the applications of the hydrogen peroxide whole-cell biological sensor, by the bacterium solution of KT2440OX bacterial strain
It is added in sample to be tested, reads the relative luminous intensity of sample, whether contain according in the relative luminous intensity judgement sample of sample
There is H2O2Or the H in calculating sample2O2Concentration.
In an embodiment of the invention, the H of known concentration is utilized2O2The bacterium of KT2440OX bacterial strain is added in sample
Relative luminous intensity after liquid, establishes H2O2Relationship between concentration and relative luminous intensity recycles the relative luminous of sample strong
Degree calculates the H in sample2O2Concentration.
In an embodiment of the invention, within the scope of hydrogen peroxide concentration 0.05mM-2mM, the hydrogen peroxide is complete
Cell biological sensor can carry out quantitative analysis to it.
In an embodiment of the invention, the luminous intensity of biosensor is with addition H2O2Amount increase and increase.
In an embodiment of the invention, the sample to be tested may include solid food sample and liquid food sample
Product, liquid food sample include plain chocolate etc.,
When the sample to be tested is solid food sample, the residual peroxide in solid food sample can be determined
Property detection；
When the sample to be tested is liquid food sample, the residual peroxide in liquid food sample can be determined
Amount detection.
In an embodiment of the invention, using microplate reader (such as selection Synergy 2, microplate
Reader, BioTek) read sample relative luminous intensity.
In an embodiment of the invention, application method specifically includes the following steps:
1), biosensor KT2440OX is incubated overnight in the LB culture medium for being added to tetracycline (50 μ g/mL), until
OD600For 0.8-1.2, preferably 1；
2), by the H of normal concentration2O2Solution and 100 μ L of sample to be tested are separately added into 96 orifice plates；
3), 100 μ L sensor bacterium solutions are separately added into above-mentioned 96 orifice plate to be measured；
4) relative luminous intensity of sample, is read；
5), data processing utilizes the H of known concentration2O2Standard curve is constructed, for calculating the H in unknown sample2O2It is dense
Degree.
In an embodiment of the invention, hydrogen peroxide whole-cell biological is added in the sample containing hydrogen peroxide to pass
After sensor reacts two hours, the H of various concentration2O2Relationship between relative luminous intensity is fitted mapping, and obtains phase
It is as follows to close relation formula:
Y=-17517*exp (- x/1168) -2222*exp (- x/40)+20450
R2=0.97331
Wherein, x be hydrogen peroxide concentration, unit be μM, y is relative luminous intensity.
Compared with prior art, in wild strain KT2440, ahpC gene encodes an alkyl hydroperoxide reductases,
When bacterial strain faces the murder by poisoning of hydrogen peroxide, will great expression, thus by hydrogen-peroxide reduction, and detoxify.This bio-sensing
The expression regulation system that ahpC gene is expressed in wild strain KT2440 is utilized in device, by the promoter of ahpC gene and report base
Because of series connection, when the expression regulation system detection of the middle ahpC gene of bacterial strain KT2440 to hydrogen peroxide there are when, inducing
While aphC gene expression, also induction of the expression of reporter gene lux, to be detected.
The present invention can establish science, accurate, quickly detection content of hydrogen peroxide method, especially suitable for directly detecting sample
Remaining hydrogen peroxide in product formulates corresponding examination criteria for national correlation department, provides some opinions and help, be one
The detection method of a very promising residual quantities of hydrogen peroxide.
Detailed description of the invention
Fig. 1 is the profile information and multiple cloning sites sequence of pCMgfp-lacZ plasmid.The plasmid is document Zhang WM,
Zhang JJ,Jiang X,Chao HJ,Zhou NY.2015.Transcriptional activation of multiple
operons involved in para-nitrophenol degradation by Pseudomonas sp strain
The plasmid announced in WBC-3.Appl Environ Microbiol 81:220-230..
Fig. 2 is the profile information and multiple cloning sites sequence of pCM-lux plasmid.Lux gene is is connected into Fig. 1 institute by the plasmid
Obtained by the plasmid shown.
Fig. 3 is the profile information and multiple cloning sites sequence of pCM-lux-PahpC plasmid.Wherein the sequence of dash area is
The sequence of the promoter PahpC of ahpC gene.The plasmid is to be connected into PahpC obtained by plasmid shown in Fig. 2.
Fig. 4 is biosensor to various concentration H2O2Response diagram.
Fig. 5 is two hours relative luminous intensities and H2O2Relational graph between concentration.
Fig. 6 is the qualitative detection of residual quantities of hydrogen peroxide in chicken claw with pickled peppers.Chicken feet H in right box2O2Processing, left box chicken feet
It does not handle.First three photo is biosensor in A.0 minute, B.30 minute and the C.1 bioluminescence in hour.D. white light is used
It takes pictures.
Fig. 7 is this biosensor to H in water, milk, Yoghourt and cola2O2Response, including Fig. 7 (a), Fig. 7 (b), figure
7 (c), Fig. 7 (d).Fig. 7 (a) KT2440OX is to H in water2O2The bioluminescence of series of concentrations.Fig. 7 (b) KT2440OX is to plain chocolate
Middle H2O2The bioluminescence of series of concentrations.Fig. 7 (c) KT2440OX is to H in yoghurt2O2The bioluminescence of series of concentrations.Fig. 7 (d)
KT2440OX is to H in yoghurt2O2The bioluminescence of series of concentrations.
Specific embodiment
The present invention is described in detail with specific embodiment below in conjunction with the accompanying drawings.
Embodiment 1
Use reagent: Phanta Super-Fidelity is only praised in Novoprotein recombination kit NR005-01A, promise
DNA Polymerase, Quan Shijin 2xEasytaq, NEB BamHI restriction endonuclease.
A kind of preparation method based on pseudomonad and lux bioluminescence gene hydrogen peroxide whole-cell biological sensor, including
Following steps:
(1) pCM-lux carrier is constructed:
The connection of lux reporter gene is entered into pCMgfp-lacZ plasmid using the method for homologous recombination, building pCM-lux is carried
Body；Expanding primer used by reporter gene is
Lux gene forward direction amplimer:
cagaaagcatgccggggatccCTCGAGCTGCAGACTAGTAGGCTTGGAGGATACGTA
TGAC
The reversed amplimer of lux gene:
cgtaatcatggtcatggatcTCAACTATCAAACGCTTCGGTTAA
Wherein small letter underlined sequences are the homology arm sequence on carrier, and tilted capital letter is more to be added by primer
Cloning site sequence.
Wherein, pCMgfp-lacZ is known plasmid, in document Zhang WM, Zhang JJ, Jiang X, Chao HJ,
Zhou NY.2015.Transcriptional activation of multiple operons involved in para-
nitrophenol degradation by Pseudomonas sp strain WBC-3.Appl Environ Microbiol
It is once had been reported that in 81:220-230..The profile information and multiple cloning sites sequence of pCMgfp-lacZ plasmid are shown in Fig. 1.pCM-lux
The profile information and multiple cloning sites sequence of plasmid are shown in Fig. 2.
(2) pCM-lux-PahpC plasmid is constructed:
Using the method for homologous recombination by ahpC gene in pseudomonas putida Pseudomonas putida KT2440
Promoter PahpC connection enters pCM-lux plasmid, constructs pCM-lux-PahpC plasmid；
Wherein, pseudomonas putida Pseudomonas putida KT2440 is known bacterial strain, in document Song Y,
Hahn T,Thompson IP,Mason TJ,Preston GM,Li G,Paniwnyk L,Huang
WE.2007.Ultrasound-mediated DNA transfer for bacteria.Nucleic Acids Res 35:
E129. there is disclosure in, the promoter PahpC of ahpC gene is in pseudomonas putida Pseudomonas putida KT2440
Known structure.
The primer used when expanding promoter are as follows:
The positive amplimer of PahpC promoter
ahpC ins cagaaagcatgccggggatccGAGCCCCTCCTTGAATGTCG
The reversed amplimer of PahpC promoter
ahpC ina agtctgcagctcgagggatccTCATGGGGTTGGAATCAGTTG。
PCM-lux-PahpC plasmid sequence is as shown in SEQ ID NO.1.The profile information of pCM-lux-PahpC plasmid and
Multiple cloning sites sequence is shown in Fig. 3.
(3) by after the pCM-lux-PahpC carrier built sequencing confirmation, it is false single that stench is transferred to using Ultrasonic transformation method
In born of the same parents bacterium Pseudomonas putida KT2440, the Pseudomonas putida KT2440 bacterial strain is disclosed in one kind
Bacterial strain, in document Song Y, Hahn T, Thompson IP, Mason TJ, Preston GM, Li G, Paniwnyk L,
Huang WE.2007.Ultrasound-mediated DNA transfer for bacteria.Nucleic Acids Res
There is disclosure in 35:e129..Bacterial strain containing pCM-lux-PahpC carrier is referred to as KT2440OX bacterial strain, and as hydrogen peroxide is complete
Cell biological sensor.
The pCM-lux carrier is used for through chemiluminescence detection promoter activity, which overcome document Zhang WM,
Zhang JJ,Jiang X,Chao HJ,Zhou NY.2015.Transcriptional activation of multiple
operons involved in para-nitrophenol degradation by Pseudomonas sp strain
PCMgfp-lacZ carrier can only utilize lacZ galactoside in WBC-3.Appl Environ Microbiol 81:220-230.
Enzymatic activity detects the limitation of substrate to be measured as reporting system, realizes the big Reporter System of lux-lacZ-xylE tri-
It connects (wherein, the two genes of lacZ-xylE are already existing in pCMgfp-lacZ plasmid, and lux connection is entered),
Great convenience is provided for follow-up signal detection.
In addition, being directed to document Zhang WM, Zhang JJ, Jiang X, Chao HJ, Zhou
NY.2015.Transcriptional activation of multiple operons involved in para-
nitrophenol degradation by Pseudomonas sp strain WBC-3.Appl Environ Microbiol
PCMgfp-lacZ carrier only has the two restriction enzyme sites of BamHI and SphI in 81:220-230., is easy to appear and is difficult to choose enzyme
The situation of enzyme site, the new support of building increase the (digestion of these enzymes of the multiple cloning sites of BamHI, XhoI, PstI, SpeI
Site is located on the primer expanded when PCR, sees the Italic capitals letter in lux gene forward direction amplimer), it is mentioned for subsequent clone
Convenience is supplied.
Embodiment 2
The present embodiment provides the applications of hydrogen peroxide whole-cell biological sensor described in embodiment 1, by KT2440OX
The bacterium solution of bacterial strain is added in hydrogen peroxide sample to be measured, reads the relative luminous intensity of sample, strong according to the relative luminous of sample
Degree calculates the H in sample2O2Concentration.
Application method specifically includes the following steps:
1), biosensor KT2440OX is incubated overnight in the LB culture medium for being added to tetracycline (50 μ g/mL), until
OD600For 0.8-1.2, preferably 1；
2), by the H of normal concentration2O2Solution and 100 μ L of sample to be tested are added in 96 orifice plates；
3), 100 μ L sensor bacterium solutions are added in 96 orifice plate to be measured；
4), using microplate reader (Synergy 2, microplate reader, BioTek) read sample luminous intensity and
OD600Numerical value；
5), data processing utilizes the H of known concentration2O2Standard curve is constructed, for calculating the H in unknown sample2O2It is dense
Degree.
The biosensor KT2440OX that this research obtains has good response to the hydrogen peroxide of various concentration, in dioxygen
Quantitative analysis can be carried out to it within the scope of water concentration 0.05mM-2mM.As shown in figure 4, passing through a period of time (0-4 hours)
Reaction, the luminous intensity of biosensor is with addition H2O2Amount increase and increase.
In order to preferably indicate luminous intensity and H2O2Relationship between concentration.We intercept the sample containing hydrogen peroxide
Middle addition hydrogen peroxide whole-cell biological is after sensor response two hours, the H of various concentration2O2Between relative luminous intensity
Relationship is fitted mapping, as shown in figure 5, and to obtain correlativity formula as follows:
Y=-17517*exp (- x/1168) -2222*exp (- x/40)+20450
R2=0.97331
Wherein, x be hydrogen peroxide concentration, unit be μM, y is relative luminous intensity.
Embodiment 3
Originally it is embodied as biosensor detection solid food residual peroxide of the present invention
It is detection background with Chicken Feet with Pickled Peppers to determine whether biosensor is effective to solid food sample.
Analytical procedure is as follows:
A. 500 milliliters of water are added in two different beakers.
B. one of that 5 milliliter of 3% hydrogen peroxide is added.
C. 2 Chicken Feet with Pickled Peppers are respectively put into beaker.
D. two samples are impregnated into 16h at 30 DEG C.
E. two chicken feets are cleaned three times.
F. 800 milliliters of KT2440OX biosensors are cultivated to stay overnight.
G. it melts 1% agar of 200ml and is incubated at 45 DEG C.
H. 1% agar is added in biosensor culture medium and quickly stirring (is likely to occur a small amount of agar block, but not shadow
Ring subsequent detection).
I. suspension is divided into two parts, respectively by H2O2Processed chicken leg and untreated chicken leg are separately added into
Biosensor suspension.
J. with the image of Bio-Rad-ChemidocTM-MP imaging system observation biosensor.
In order to explore H2O2The application of biosensor, by taking Chinese traditional food Chicken Feet with Pickled Peppers as an example, it is contemplated that it is typical
Appearance.In addition, addition hydrogen peroxide is common in the production process of the food such as Chicken Feet with Pickled Peppers.As shown in fig. 6, working as chicken
When pawl is added in biosensor suspension, its appearance is dark, because itself does not shine.With pushing away for time
It moves, by H2O2The chicken feet (right crus of diaphragm in figure) of processing gradually brightens.
This research as a result, it was confirmed that biosensor KT2440OX can be used to detect peroxidating in solid food sample
Hydrogen residual.
Embodiment 4
Residual peroxide in this biosensor detection liquid food is utilized in the present embodiment
Steps are as follows:
1), biosensor KT2440OX is incubated overnight in the LB culture medium for being added to tetracycline (50 μ g/mL), until
OD600 is 0.8-1.2, preferably 1；
2), by the H of known concentration2O2, it is added in water, milk, Yoghourt, cola etc., configures respective standard solution, it will be each
The 100 μ L of standard solution of concentration is added in 96 orifice plates；
3), 100 μ L sensor bacterium solutions are added in above-mentioned 96 orifice plate to be measured；
4) luminous intensity of sample, is read using microplate reader (Synergy 2, microplate reader, BioTek)；
5), data processing utilizes the H of known concentration2O2Standard curve is constructed, for calculating the H in unknown sample2O2It is dense
Degree.
To determine that can biosensor KT2440OX can be used for detecting the hydrogen peroxide in beverage.This research is attempted in ox
This biosensor is tested in milk, Yoghourt and cola to H2O2Response.Since the absorptivity of these sample light is high, we
It was originally thought that the hope of measurement bioluminescence is little.Since the light absorption of sample is higher, biosensor cell is not considered
OD value.Therefore, the data of the present embodiment are shown as bioluminescence rather than opposite bioluminescence, as shown in Figure 7.With absolute
Light absorption is come to the H in water sample2O2When being measured, biosensor KT2440OX still has preferable response.Enable us surprised
It is that it is more preferable to the effect of milk sample.The high light absorption of milk does not influence to H2O2The detection of concentration, internal nutritional ingredient
The luminescent properties of biosensor can be improved.But for Yoghourt, biosensor cannot work.Biosensor is to acid
The bioluminescence ability of various concentration of hydrogen peroxide is weaker in milk.This may be because are as follows: the high concentration lactic acid in a. Yoghourt is to life
Object sensor is toxic.B. a large amount of viable bacteria inhibits the activity of biosensor.C. catalase present in Yoghourt viable bacteria
The hydrogen peroxide of addition may be degraded.For laughable sample, concentration of hydrogen peroxide is higher, and bioluminescence intensity is higher.But believe
Number intensity, stability and correlation are undesirable.In addition to this, laughable production process is usually extensive and standardized.It is raw
Object sensor pair and laughable similar sample only have and certain have reference value.In conclusion biosensor
KT2440OX can detecte the hydrogen peroxide in fluid sample especially plain chocolate.
The above description of the embodiments is intended to facilitate ordinary skill in the art to understand and use the invention.
Person skilled in the art obviously easily can make various modifications to these embodiments, and described herein general
Principle is applied in other embodiments without having to go through creative labor.Therefore, the present invention is not limited to the above embodiments, ability
Field technique personnel announcement according to the present invention, improvement and modification made without departing from the scope of the present invention all should be of the invention
Within protection scope.
Sequence table
<110>Shanghai He Sen Biotechnology Co., Ltd
<120>a kind of hydrogen peroxide whole-cell biological sensor and its preparation and application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 17783
<212> DNA
<213>pseudomonas putida (Pseudomonas putida)
<400> 1
gacggatctt ttccgctgca taaccctgct tcggggtcat tatagcgatt ttttcggtat 60
atccatcctt tttcgcacga tatacaggat tttgccaaag ggttcgtgta gactttcctt 120
ggtgtatcca acggcgtcag ccgggcagga taggtgaagt aggcccaccc gcgagcgggt 180
gttccttctt cactgtccct tattcgcacc tggcggtgct caacgggaat cctgctctgc 240
gaggctggcc ggctaccgcc ggcgtaacag atgagggcaa gcggatggct gatgaaacca 300
agccaaccag gaagggcagc ccacctatca aggtgtactg ccttccagac gaacgaagag 360
cgattgagga aaaggcggcg gcggccggca tgagcctgta ggcctacctg ctggccgtcg 420
gccagggcta caaaatcacg ggcgtcgtgg actatgagca cgtccgcgag ctggcccgca 480
tcaatggcga cctgggccgc ctgggcggcc tgctgaaact ctggctcacc gacgacccgc 540
gcacggcgcg gttcggtgat gccacgatcc tcgccctgct ggcgaagatc gaagagaagc 600
aggacgagct tggcaaggtc atgatgggcg tggtccgccc gagggcagag ccatgacttt 660
tttagccgct aaaacggccg gggggtgcgc gtgattgcca agcacgtccc catgcgctcc 720
atcaagaaga gcgacttcgc ggagctggta ttcgtgcagg gcaagattcg gaataccaag 780
tacgagaagg acggccagac ggtctacggg accgacttca ttgccgataa ggtggattat 840
ctggacacca aggcaccagg cgggtcaaat caggaataag ggcacattgc cccggcgtga 900
gtcggggcaa tcccgcaagg agggtgaatg aatcggacgt ttgaccggaa ggcatacagg 960
caagaactga tcgacgcggg gttttccgcc gaggatgccg aaaccatcgc aagccgcacc 1020
gtcatgcgtg cgccccgcga aaccttccag tccgtcggct cgatggtcca gcaagctacg 1080
gccaagatcg agcgcgacag cgtgcaactg gctccccctg ccctgcccgc gccatcggcc 1140
gccgtggagc gttcgcgtcg tctcgaacag gaggcggcag gtttggcgaa gtcgatgacc 1200
atcgacacgc gaggaactat gacgaccaag aagcgaaaaa ccgccggcga ggacctggca 1260
aaacaggtca gcgaggccaa gcaggccgcg ttgctgaaac acacgaagca gcagatcaag 1320
gaaatgcagc tttccttgtt cgatattgcg ccgtggccgg acacgatgcg agcgatgcca 1380
aacgacacgg cccgctctgc cctgttcacc acgcgcaaca agaaaatccc gcgcgaggcg 1440
ctgcaaaaca aggtcatttt ccacgtcaac aaggacgtga agatcaccta caccggcgtc 1500
gagctgcggg ccgacgatga cgaactggtg tggcagcagg tgttggagta cgcgaagcgc 1560
acccctatcg gcgagccgat caccttcacg ttctacgagc tttgccagga cctgggctgg 1620
tcgatcaatg gccggtatta cacgaaggcc gaggaatgcc tgtcgcgcct acaggcgacg 1680
gcgatgggct tcacgtccga ccgcgttggg cacctggaat cggtgtcgct gctgcaccgc 1740
ttccgcgtcc tggaccgtgg caagaaaacg tcccgttgcc aggtcctgat cgacgaggaa 1800
atcgtcgtgc tgtttgctgg cgaccactac acgaaattca tatgggagaa gtaccgcaag 1860
ctgtcgccga cggcccgacg gatgttcgac tatttcagct cgcaccggga gccgtacccg 1920
ctcaagctgg aaaccttccg cctcatgtgc ggatcggatt ccacccgcgt gaagaagtgg 1980
cgcgagcagg tcggcgaagc ctgcgaagag ttgcgaggca gcggcctggt ggaacacgcc 2040
tgggtcaatg atgacctggt gcattgcaaa cgctagggcc ttgtggggtc agttccggct 2100
gggggttcag cagccagcgc tttactggca tttcaggaac aagcgggcac tgctcgacgc 2160
acttgcttcg ctcagtatcg ctcgggacgc acggcgcgct ctacgaactg ccgataaaca 2220
gaggattaaa attgacaatt ctagggcgcg tatagcttgc cggaagtcgc cttgacccgc 2280
atggcatagg cctatcgttt ccacgatcag cgatcggctc gttgccctgc gccgctccaa 2340
agcccgcgac gcagcgccgg caggcagagc aagtagaggg cagcgcctgc aatccatgcc 2400
cacccgttcc acgttgttat agaagccgca tagatcgccg tgaagaggag gggtccgacg 2460
atcgaggtca ggctggtgag cgccgccagt gagccttgca gctgcccctg acgttcctca 2520
tccacctgcc tggacaacat tgcttgcagc gccggcattc cgatgccacc cgaagcaagc 2580
aggaccatga tcgggaacgc catccatccc cgtgtcgcga aggcaagcag gatgtagcct 2640
gtgccgtcgg caatcattcc gagcatgagt gcccgccttt cgccgagccg ggcggctaca 2700
gggccggtga tcattgcctg ggcgagtgaa tgcagaatgc caaatgcggc aagcgaaatg 2760
ccgatcgtgg tcgcgtccca gtgaaagcga tcctcgccga aaatgaccca aagcgcggcc 2820
ggcacctgtc cgacaagttg catgatgaag aagaccgcca tcagggcggc gacgacggtc 2880
atgccccggg cccaccggaa cgaagcgagc gggttgagag cctcccggcg taacggccgg 2940
cgttcgcctt tgtgcgactc cggcaaaagg aaacagcccg tcaggaaatt gaggccgttc 3000
aaggctgccg cggcgaagaa cggagcgtgg ggggagaaac cgcccatcag cccaccgagc 3060
acaggtcccg cgaccatccc gaacccgaaa caggcgctca tgaagccgaa gtgccgcgcg 3120
cgctcatcgc catcagtgat atcggcaata taagcgccgg ctaccgcccc agtcgccccg 3180
gtgatgccgg ccacgatccg cccgatatag agaacccaaa ggaaaggcgc tgtcgccatg 3240
atggcgtagt cgacagtggc gccggccagc gagacgagca agattggccg ccgcccgaaa 3300
cgatccgaca gcgcgcccag cacaggtgcg caggcaaatt gcaccaacgc atacagcgcc 3360
agcagaatgc catagtgggc ggtgacgtcg ttcgagtgaa ccagatcgcg caggaggccc 3420
ggcagcaccg gcataatcag gccgatgccg acagcgtcga gcgcgacagt gctcagaatt 3480
acgatcaggg gtatgttggg tttcacgtct ggcctccgga ccagcctccg ctggtccgat 3540
tgaacgcgcg gattctttat cactgataag ttggtggaca tattatgttt atcagtgata 3600
aagtgtcaat catgacaaag ttgcagccga atacagtgat ccgtgccgcc ctggacctgt 3660
tgaacgaggt cggcgtagac ggtctgacga cacgcaaact ggcggaacgg ttgggggttc 3720
agcagccggc gctttactgg cacttcagga acaagcgggc gctgctcgac gcactggccg 3780
aagccatgct ggcggagaat catacgcatt cggtgccgag agccgacgac gactggcgct 3840
catttctgat cgggaatgcc cgcagcttca ggcaggcgct gctcgcctac cgcgatggcg 3900
cgcgcatcca tgccggcacg cgaccgggcg caccgcagat ggaaacggcc gacgcgcagc 3960
ttcgcttcct ctgcgaggcg ggtttttcgg ccggggacgc cgtcaatgcg ctgatgacaa 4020
tcagctactt cactgttggg gccgtgcttg aggagcaggc cggcgacagc gatgccggcg 4080
agcgcggcgg caccgttgaa caggctccgc tctcgccgct gttgcgggcc gcgatagacg 4140
ccttcgacga agccggtccg gacgcagcgt tcgagcaggg actcgcggtg attgtcgatg 4200
gattggcgaa aaggaggctc gttgtcagga acgttgaagg accgagaaag ggtgctaaga 4260
aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct 4320
cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac 4380
agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt 4440
tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca 4500
ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgcca 4560
ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt 4620
acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt 4680
ttcccagtca cgacgttgta aaacgacggc cagtgaattt ttcagcctga tacagattaa 4740
atcagaacgc agaagcggtc tgataaaaca gaatttgcct ggcggcagta gcgcggtggt 4800
cccacctgac cccatgccga actcagaagt gaaacgccgt agcgccgatg gtagtgtggg 4860
gtctccccat gcgagagtag ggaactgcca ggcatcaaat aaaacgaaag gctcagtcga 4920
aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa 4980
atccgccggg agcggatttg aacgttgcga agcaacggcc cggagggtgg cgggcaggac 5040
gcccgccata aactgccagg catcaaatta agcagaaggc catcctgacg gatggccttt 5100
ttgcgtttct acaaactctt cctgtcgtca tatctacaag ccatcccccc acagatacgg 5160
taaactagcc tcgtttttgc atcaggaaag cagggaattc tagattcgag ctccatgtgt 5220
aatcccagca gctgttacaa actcaagaag gaccatgtgg tctctctttt cgttgggatc 5280
tttcgaaagg gcagattgtg tggacaggta atggttgtct ggtaaaagga cagggccatc 5340
gccaattgga gtattttgtt gataatggtc tgctagttga acgcttccat cttcaatgtt 5400
gtgtctaatt ttgaagttaa ctttgattcc attcttttgt ttgtctgcca tgatgtatac 5460
attgtgtgag ttatagttgt attccaattt gtgtccaaga atgtttccat cttctttaaa 5520
atcaatacct tttaactcga ttctattaac aagggtatca ccttcaaact tgacttcagc 5580
acgtgtcttg tagttcccgt catctttgaa aaatatagtt ctttcctgta cataaccttc 5640
gggcatggca ctcttgaaaa agtcatgctg tctcatatga tctgggtatc ttgaaaagca 5700
ttgaacacca taagtcagag tagtgacaag tgttggccat ggaacaggta gttttccagt 5760
agtgcaaata aatttaaggg taagttttcc gtatgttgca tcaccttcac cctctccact 5820
gacagaaagc atgccgggga tccgagcccc tccttgaatg tcgacgttgc aaggcaacag 5880
tgatccgaca tggcggcttg gcagccgtca gaggggcccg gtatcacatg ggcttgagcg 5940
tagacccgtt tgcccgtgat accgaatggg gagctgttac atttctttgc cagtgcagag 6000
ttcaggtccg caatcctttg ctcagcccgt tcccttctat taaataaccc cagttatagc 6060
caaaaccaat cgaaagcatg agctttgcca atgagccttc agcgggatga ttagttagga 6120
ttctatccat caactgattc caaccccatg aggatccctc gagctgcaga ctagtaggct 6180
tggaggatac gtatgactaa aaaaatttca ttcattatta acggccaggt tgaaatcttt 6240
cccgaaagtg atgatttagt gcaatccatt aattttggtg ataatagtgt ttacctgcca 6300
atattgaatg actctcatgt aaaaaacatt attgattgta atggaaataa cgaattacgg 6360
ttgcataaca ttgtcaattt tctctatacg gtagggcaaa gatggaaaaa tgaagaatac 6420
tcaagacgca ggacatacat tcgtgactta aaaaaatata tgggatattc agaagaaatg 6480
gctaagctag aggccaattg gatatctatg attttatgtt ctaaaggcgg cctttatgat 6540
gttgtagaaa atgaacttgg ttctcgccat atcatggatg aatggctacc tcaggatgaa 6600
agttatgttc gggcttttcc gaaaggtaaa tctgtacatc tgttggcagg taatgttcca 6660
ttatctggga tcatgtctat attacgcgca attttaacta agaatcagtg tattataaaa 6720
acatcgtcaa ccgatccttt taccgctaat gcattagcgt taagttttat tgatgtagac 6780
cctaatcatc cgataacgcg ctctttatct gttatatatt ggccccacca aggtgataca 6840
tcactcgcaa aagaaattat gcgacatgcg gatgttattg tcgcttgggg agggccagat 6900
gcgattaatt gggcggtaga gcatgcgcca tcttatgctg atgtgattaa atttggttct 6960
aaaaagagtc tttgcattat cgataatcct gttgatttga cgtccgcagc gacaggtgcg 7020
gctcatgatg tttgttttta cgatcagcga gcttgttttt ctgcccaaaa catatattac 7080
atgggaaatc attatgagga atttaagtta gcgttgatag aaaaacttaa tctatatgcg 7140
catatattac cgaatgccaa aaaagatttt gatgaaaagg cggcctattc tttagttcaa 7200
aaagaaagct tgtttgctgg attaaaagta gaggtggata ttcatcaacg ttggatgatt 7260
attgagtcaa atgcaggtgt ggaatttaat caaccacttg gcagatgtgt gtaccttcat 7320
cacgtcgata atattgagca aatattgcct tatgttcaaa aaaataagac gcaaaccata 7380
tctatttttc cttgggagtc atcatttaaa tatcgagatg cgttagcatt aaaaggtgcg 7440
gaaaggattg tagaagcagg aatgaataac atatttcgag ttggtggatc tcatgacgga 7500
atgagaccgt tgcaacgatt agtgacatat atttctcatg aaaggccatc taactatacg 7560
gctaaggatg ttgcggttga aatagaacag actcgattcc tggaagaaga taagttcctt 7620
gtatttgtcc cataataggt aaaagtatgg aaaatgaatc aaaatataaa accatcgacc 7680
acgttatttg tgttgaagga aataaaaaaa ttcatgtttg ggaaacgctg ccagaagaaa 7740
acagcccaaa gagaaagaat gccattatta ttgcgtctgg ttttgcccgc aggatggatc 7800
attttgctgg tctggcggaa tatttatcgc ggaatggatt tcatgtgatc cgctatgatt 7860
cgcttcacca cgttggattg agttcaggga caattgatga atttacaatg tctataggaa 7920
agcagagctt gttagcagtg gttgattggt taactacacg aaaaataaat aacttcggta 7980
tgttggcttc aagcttatct gcgcggatag cttatgcaag cctatctgaa atcaatgctt 8040
cgtttttaat caccgcagtc ggtgttgtta acttaagata ttctcttgaa agagctttag 8100
ggtttgatta tctcagtcta cccattaatg aattgccgga taatctagat tttgaaggcc 8160
ataaattggg tgctgaagtc tttgcgagag attgtcttga ttttggttgg gaagatttag 8220
cttctacaat taataacatg atgtatcttg atataccgtt tattgctttt actgcaaata 8280
acgataattg ggtcaagcaa gatgaagtta tcacattgtt atcaaatatt cgtagtaatc 8340
gatgcaagat atattctttg ttaggaagtt cgcatgactt gagtgaaaat ttagtggtcc 8400
tgcgcaattt ttatcaatcg gttacgaaag ccgctatcgc gatggataat gatcatctgg 8460
atattgatgt tgatattact gaaccgtcat ttgaacattt aactattgcg acagtcaatg 8520
aacgccgaat gagaattgag attgaaaatc aagcaatttc tctgtcttaa aatctattga 8580
gatattctat cactcaaata gcaatataag gactctctat gaaatttgga aactttttgc 8640
ttacatacca acctccccaa ttttctcaaa cagaggtaat gaaacgtttg gttaaattag 8700
gtcgcatctc tgaggagtgt ggttttgata ccgtatggtt actggagcat catttcacgg 8760
agtttggttt gcttggtaac ccttatgtcg ctgctgcata tttacttggc gcgactaaaa 8820
aattgaatgt aggaactgcc gctattgttc ttcccacagc ccatccagta cgccaacttg 8880
aagatgtgaa tttattggat caaatgtcaa aaggacgatt tcggtttggt atttgccgag 8940
ggctttacaa caaggacttt cgcgtattcg gcacagatat gaataacagt cgcgccttag 9000
cggaatgctg gtacgggctg ataaagaatg gcatgacaga gggatatatg gaagctgata 9060
atgaacatat caagttccat aaggtaaaag taaaccccgc ggcgtatagc agaggtggcg 9120
caccggttta tgtggtggct gaatcagctt cgacgactga gtgggctgct caatttggcc 9180
taccgatgat attaagttgg attataaata ctaacgaaaa gaaagcacaa cttgagcttt 9240
ataatgaagt ggctcaagaa tatgggcacg atattcataa tatcgaccat tgcttatcat 9300
atataacatc tgtagatcat gactcaatta aagcgaaaga gatttgccgg aaatttctgg 9360
ggcattggta tgattcttat gtgaatgcta cgactatttt tgatgattca gaccaaacaa 9420
gaggttatga tttcaataaa gggcagtggc gtgactttgt attaaaagga cataaagata 9480
ctaatcgccg tattgattac agttacgaaa tcaatcccgt gggaacgccg caggaatgta 9540
ttgacataat tcaaaaagac attgatgcta caggaatatc aaatatttgt tgtggatttg 9600
aagctaatgg aacagtagac gaaattattg cttccatgaa gctcttccag tctgatgtca 9660
tgccatttct taaagaaaaa caacgttcgc tattatatta gctaaggaga aagaaatgaa 9720
atttggattg ttcttcctta acttcatcaa ttcaacaact gttcaagaac aaagtatagt 9780
tcgcatgcag gaaataacgg agtatgttga taagttgaat tttgaacaga ttttagtgta 9840
tgaaaatcat ttttcagata atggtgttgt cggcgctcct ctgactgttt ctggttttct 9900
gctcggttta acagagaaaa ttaaaattgg ttcattaaat cacatcatta caactcatca 9960
tcctgtcgcc atagcggagg aagcttgctt attggatcag ttaagtgaag ggagatttat 10020
tttagggttt agtgattgcg aaaaaaaaga tgaaatgcat ttttttaatc gcccggttga 10080
atatcaacag caactatttg aagagtgtta tgaaatcatt aacgatgctt taacaacagg 10140
ctattgtaat ccagataacg atttttatag cttccctaaa atatctgtaa atccccatgc 10200
ttatacgcca ggcggacctc ggaaatatgt aacagcaacc agtcatcata ttgttgagtg 10260
ggcggccaaa aaaggtattc ctctcatctt taagtgggat gattctaatg atgttagata 10320
tgaatatgct gaaagatata aagccgttgc ggataaatat gacgttgacc tatcagagat 10380
agaccatcag ttaatgatat tagttaacta taacgaagat agtaataaag ctaaacaaga 10440
gacgcgtgca tttattagtg attatgttct tgaaatgcac cctaatgaaa atttcgaaaa 10500
taaacttgaa gaaataattg cagaaaacgc tgtcggaaat tatacggagt gtataactgc 10560
ggctaagttg gcaattgaaa agtgtggtgc gaaaagtgta ttgctgtcct ttgaaccaat 10620
gaatgatttg atgagccaaa aaaatgtaat caatattgtt gatgataata ttaagaagta 10680
ccacatggaa tatacctaat agatttcgag ttgcagcgag gcggcaagtg aacgaatccc 10740
caggagcata gataactatg tgactggggt gagtgaaagc agccaacaaa gcagcagctt 10800
gaaagatgaa gggtataaaa gagtatgaca gcagtgctgc catactttct aatattatct 10860
tgaggagtaa aacaggtatg acttcatatg ttgataaaca agaaattaca gcaagctcag 10920
aaattgatga tttgattttt tcgagcgatc cattagtgtg gtcttacgac gagcaggaaa 10980
aaatcagaaa gaaacttgtg cttgatgcat ttcgtaatca ttataaacat tgtcgagaat 11040
atcgtcacta ctgtcaggca cacaaagtag atgacaatat tacggaaatt gatgacatac 11100
ctgtattccc aacatcggtt tttaagttta ctcgcttatt aacttctcag gaaaacgaga 11160
ttgaaagttg gtttaccagt agcggcacga atggtttaaa aagtcaggtg gcgcgtgaca 11220
gattaagtat tgagagactc ttaggctctg tgagttatgg catgaaatat gttggtagtt 11280
ggtttgatca tcaaatagaa ttagtcaatt tgggaccaga tagatttaat gctcataata 11340
tttggtttaa atatgttatg agtttggtgg aattgttata tcctacgaca tttaccgtaa 11400
cagaagaacg aatagatttt gttaaaacat tgaatagtct tgaacgaata aaaaatcaag 11460
ggaaagatct ttgtcttatt ggttcgccat actttattta tttactctgc cattatatga 11520
aagataaaaa aatctcattt tctggagata aaagccttta tatcataacc ggaggcggct 11580
ggaaaagtta cgaaaaagaa tctctgaaac gtgatgattt caatcatctt ttatttgata 11640
ctttcaatct cagtgatatt agtcagatcc gagatatatt taatcaagtt gaactcaaca 11700
cttgtttctt tgaggatgaa atgcagcgta aacatgttcc gccgtgggta tatgcgcgag 11760
cgcttgatcc tgaaacgttg aaacctgtac ctgatggaac gccggggttg atgagttata 11820
tggatgcgtc agcaaccagt tatccagcat ttattgttac cgatgatgtc gggataatta 11880
gcagagaata tggtaagtat cccggcgtgc tcgttgaaat tttacgtcgc gtcaatacga 11940
ggacgcagaa agggtgtgct ttaagcttaa ccgaagcgtt tgatagttga gatccatgac 12000
catgattacg gattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt 12060
tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga 12120
ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgctttgc 12180
ctggtttccg gcaccagaag cggtgccgga aagctggctg gagtgcgatc ttcctgaggc 12240
cgatactgtc gtcgtcccct caaactggca gatgcacggt tacgatgcgc ccatctacac 12300
caacgtgacc tatcccatta cggtcaatcc gccgtttgtt cccacggaga atccgacggg 12360
ttgttactcg ctcacattta atgttgatga aagctggcta caggaaggcc agacgcgaat 12420
tatttttgat ggcgttaact cggcgtttca tctgtggtgc aacgggcgct gggtcggtta 12480
cggccaggac agtcgtttgc cgtctgaatt tgacctgagc gcatttttac gcgccggaga 12540
aaaccgcctc gcggtgatgg tgctgcgctg gagtgacggc agttatctgg aagatcagga 12600
tatgtggcgg atgagcggca ttttccgtga cgtctcgttg ctgcataaac cgactacaca 12660
aatcagcgat ttccatgttg ccactcgctt taatgatgat ttcagccgcg ctgtactgga 12720
ggctgaagtt cagatgtgcg gcgagttgcg tgactaccta cgggtaacag tttctttatg 12780
gcagggtgaa acgcaggtcg ccagcggcac cgcgcctttc ggcggtgaaa ttatcgatga 12840
gcgtggtggt tatgccgatc gcgtcacact acgtctgaac gtcgaaaacc cgaaactgtg 12900
gagcgccgaa atcccgaatc tctatcgtgc ggtggttgaa ctgcacaccg ccgacggcac 12960
gctgattgaa gcagaagcct gcgatgtcgg tttccgcgag gtgcggattg aaaatggtct 13020
gctgctgctg aacggcaagc cgttgctgat tcgaggcgtt aaccgtcacg agcatcatcc 13080
tctgcatggt caggtcatgg atgagcagac gatggtgcag gatatcctgc tgatgaagca 13140
gaacaacttt aacgccgtgc gctgttcgca ttatccgaac catccgctgt ggtacacgct 13200
gtgcgaccgc tacggcctgt atgtggtgga tgaagccaat attgaaaccc acggcatggt 13260
gccaatgaat cgtctgaccg atgatccgcg ctggctaccg gcgatgagcg aacgcgtaac 13320
gcgaatggtg cagcgcgatc gtaatcaccc gagtgtgatc atctggtcgc tggggaatga 13380
atcaggccac ggcgctaatc acgacgcgct gtatcgctgg atcaaatctg tcgatccttc 13440
ccgcccggtg cagtatgaag gcggcggagc cgacaccacg gccaccgata ttatttgccc 13500
gatgtacgcg cgcgtggatg aagaccagcc cttcccggct gtgccgaaat ggtccatcaa 13560
aaaatggctt tcgctacctg gagagacgcg cccgctgatc ctttgcgaat acgcccacgc 13620
gatgggtaac agtcttggcg gtttcgctaa atactggcag gcgtttcgtc agtatccccg 13680
tttacagggc ggcttcgtct gggactgggt ggatcagtcg ctgattaaat atgatgaaaa 13740
cggcaacccg tggtcggctt acggcggtga ttttggcgat acgccgaacg atcgccagtt 13800
ctgtatgaac ggtctggtct ttgccgaccg cacgccgcat ccagcgctga cggaagcaaa 13860
acaccagcag cagtttttcc agttccgttt atccgggcaa accatcgaag tgaccagcga 13920
atacctgttc cgtcatagcg ataacgagct cctgcactgg atggtggcgc tggatggtaa 13980
gccgctggca agcggtgaag tgcctctgga tgtcgctcca caaggtaaac agttgattga 14040
actgcctgaa ctaccgcagc cggagagcgc cgggcaactc tggctcacag tacgcgtagt 14100
gcaaccgaac gcgaccgcat ggtcagaagc cggacacatc agcgcctggc agcagtggcg 14160
tctggctgaa aacctcagcg tgacactccc cgccgcgtcc cacgccatcc cgcatctgac 14220
caccagcgaa atggattttt gcatcgagct gggtaataag cgttggcaat ttaaccgcca 14280
gtcaggcttt ctttcacaga tgtggattgg cgataaaaaa caactgctga cgccgctgcg 14340
cgatcagttc acccgtgcac cgctggataa cgacattggc gtaagtgaag cgacccgcat 14400
tgaccctaac gcctgggtcg aacgctggaa ggcggcgggc cattaccagg ccgaagcagc 14460
gttgttgcag tgcacggcag atacacttgc tgatgcggtg ctgattacga ccgctcacgc 14520
gtggcagcat caggggaaaa ccttatttat cagccggaaa acctaccgga ttgatggtag 14580
tggtcaaatg gcgattaccg ttgatgttga agtggcgagc gatacaccgc atccggcgcg 14640
gattggcctg aactgccagc tggcgcaggt agcagagcgg gtaaactggc tcggattagg 14700
gccgcaagaa aactatcccg accgccttac tgccgcctgt tttgaccgct gggatctgcc 14760
attgtcagac atgtataccc cgtacgtctt cccgagcgaa aacggtctgc gctgcgggac 14820
gcgcgaattg aattatggcc cacaccagtg gcgcggcgac ttccagttca acatcagccg 14880
ctacagtcaa cagcaactga tggaaaccag ccatcgccat ctgctgcacg cggaagaagg 14940
cacatggctg aatatcgacg gtttccatat ggggattggt ggcgacgact cctggagccc 15000
gtcagtatcg gcggaattcc agctgagcgc cggtcgctac cattaccagt tggtctggtg 15060
tcaaaaataa aagcttcagg aggtgacgtc atgaacaaag gtgtaatgcg accgggccat 15120
gtgcagctgc gtgtactgga catgagcaag gccctggaac actacgtcga gttgctgggc 15180
ctgatcgaga tggaccgtga cgaccagggc cgtgtctatc tgaaggcttg gaccgaagtg 15240
gataagtttt ccctggtgct acgcgaggct gacgagccgg gcatggattt tatgggtttc 15300
aaggttgtgg atgaggatgc tctccggcaa ctggagcggg atctgatggc atatggctgt 15360
gccgttgagc agctacccgc aggtgaactg aacagttgtg gccggcgcgt gcgcttccag 15420
gccccctccg ggcatcactt cgagttgtat gcagacaagg aatatactgg aaagtggggt 15480
ttgaatgacg tcaatcccga ggcatggccg cgcgatctga aaggtatggc ggctgtgcgt 15540
ttcgaccacg ccctcatgta tggcgacgaa ttgccggcga cctatgacct gttcaccaag 15600
gtgctcggtt tctatctggc cgaacaggtg ctggacgaaa atggcacgcg cgtcgcccag 15660
tttctcagtc tgtcgaccaa ggcccacgac gtggccttca ttcaccatcc ggaaaaaggc 15720
cgcctccatc atgtgtcctt ccacctcgaa acctgggaag acttgcttcg cgccgccgac 15780
ctgatctcca tgaccgacac atctatcgat atcggcccaa cccgccacgg cctcactcac 15840
ggcaagacca tctacttctt cgacccgtcc ggtaaccgca acgaagtgtt ctgcggggga 15900
gattacaact acccggacca caaaccggtg acctggacca ccgaccagct gggcaaggcg 15960
atcttttacc acgaccgcat tctcaacgaa cgattcatga ccgtgctcac ctgatggagc 16020
tcccgggtac catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt 16080
tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa 16140
gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct 16200
ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc 16260
cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg 16320
tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct 16380
tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag 16440
cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga 16500
agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga 16560
agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg 16620
gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag 16680
aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag 16740
ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat 16800
gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct 16860
taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac 16920
tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa 16980
tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg 17040
gaagggccga gcgcagaagt ggtcagcttg gctgccattt ttggggtgag gccgttcgcg 17100
gccgaggggc gcagcccctg gggggatggg aggcccgcgt tagcgggccg ggagggttcg 17160
agaagggggg gcacccccct tcggcgtgcg cggtcacgcg cacagggcgc agccctggtt 17220
aaaaacaagg tttataaata ttggtttaaa agcaggttaa aagacaggtt agcggtggcc 17280
gaaaaacggg cggaaaccct tgcaaatgct ggattttctg cctgtggaca gcccctcaaa 17340
tgtcaatagg tgcgcccctc atctgtcagc actctgcccc tcaagtgtca aggatcgcgc 17400
ccctcatctg tcagtagtcg cgcccctcaa gtgtcaatac cgcagggcac ttatccccag 17460
gcttgtccac atcatctgtg ggaaactcgc gtaaaatcag gcgttttcgc cgatttgcga 17520
ggctggccag ctccacgtcg ccggccgaaa tcgagcctgc ccctcatctg tcaacgccgc 17580
gccgggtgag tcggcccctc aagtgtcaac gtccgcccct catctgtcag tgagggccaa 17640
gttttccgcg aggtatccac aacgccggcg gccgcggtgt ctcgcacacg gcttcgacgg 17700
cgtttctggc gcgtttgcag ggccatagac ggccgccagc ccagcggcga gggcaaccag 17760
cccggtgagc gtcggaaagg gtc 17783
Claims (10)
1. a kind of preparation method of hydrogen peroxide whole-cell biological sensor, which comprises the following steps:
(1) pCM-lux carrier is constructed:
The connection of lux reporter gene is entered into pCMgfp-lacZ plasmid using the method for homologous recombination, constructs pCM-lux carrier；
(2) pCM-lux-PahpC carrier is constructed:
Using the method for homologous recombination by the starting of ahpC gene in pseudomonas putida Pseudomonas putida KT2440
Sub- PahpC connection enters pCM-lux plasmid, constructs pCM-lux-PahpC carrier；
(3) by after the pCM-lux-PahpC carrier built sequencing confirmation, it is transferred to pseudomonas putida Pseudomonas
In putida KT2440, obtained strains are referred to as KT2440OX bacterial strain, as hydrogen peroxide whole-cell biological sensor.
2. a kind of preparation method of hydrogen peroxide whole-cell biological sensor according to claim 1, which is characterized in that
Step (1) expands primer used by lux reporter gene
Lux gene forward direction amplimer:
cagaaagcatgccggggatccCTCGAGCTGCAGACTAGTAGGCTTGGAGGATACGTATGAC
The reversed amplimer of lux gene:
cgtaatcatggtcatggatcTCAACTATCAAACGCTTCGGTTAA
Wherein small letter underlined sequences are the homology arm sequence on carrier, and tilted capital letter is polyclonal to be added by primer
Site sequence；
Step (2) expands the primer used when promoter are as follows:
The positive amplimer of PahpC promoter
ahpC ins cagaaagcatgccggggatccGAGCCCCTCCTTGAATGTCG
The reversed amplimer of PahpC promoter
ahpC ina agtctgcagctcgagggatccTCATGGGGTTGGAATCAGTTG。
3. the hydrogen peroxide whole-cell biological sensor that preparation method obtains according to claim 1.
4. the application of hydrogen peroxide whole-cell biological sensor as claimed in claim 3, which is characterized in that by KT2440OX bacterium
The bacterium solution of strain is added in sample to be tested, the relative luminous intensity of sample is read, according to the relative luminous intensity judgement sample of sample
In whether contain H2O2Or the H in calculating sample2O2Concentration.
5. the application of hydrogen peroxide whole-cell biological sensor according to claim 4, which is characterized in that utilize known dense
The H of degree2O2Relative luminous intensity after the bacterium solution of KT2440OX bacterial strain is added in sample, establishes H2O2Concentration is strong with relative luminous
Relationship between degree recycles the relative luminous intensity of sample to calculate the H in sample2O2Concentration.
6. the application of hydrogen peroxide whole-cell biological sensor according to claim 4, which is characterized in that dense in hydrogen peroxide
It spends within the scope of 0.05mM-2mM, the hydrogen peroxide whole-cell biological sensor can carry out quantitative analysis to it.
7. the application of hydrogen peroxide whole-cell biological sensor according to claim 4, which is characterized in that biosensor
Luminous intensity with addition H2O2Amount increase and increase.
8. the application of hydrogen peroxide whole-cell biological sensor according to claim 4, which is characterized in that described to test sample
Product include solid food sample and liquid food sample,
When the sample to be tested is solid food sample, qualitative inspection can be carried out to the residual peroxide in solid food sample
It surveys；
When the sample to be tested is liquid food sample, quantitative inspection can be carried out to the residual peroxide in liquid food sample
It surveys.
9. the application of hydrogen peroxide whole-cell biological sensor according to claim 4, which is characterized in that method include with
Lower step:
1), hydrogen peroxide whole-cell biological sensor is incubated overnight in the LB culture medium for being added to tetracycline, until OD600For
0.8-1.2；
2), by the H of normal concentration2O2Solution and 100 μ L of sample to be tested are separately added into 96 orifice plates；
3), 100 μ L sensor bacterium solutions are separately added into the hole for having been added standard solution and sample on 96 orifice plate to be measured；
4) relative luminous intensity of sample, is read；
5), data processing utilizes the H of known concentration2O2Standard curve is constructed, for calculating the H in unknown sample2O2Concentration.
10. the application of hydrogen peroxide whole-cell biological sensor according to claim 9, which is characterized in that contain peroxide
Change and hydrogen peroxide whole-cell biological sensor response is added in the sample of hydrogen after two hours, the H of various concentration2O2With relative luminous
Relationship between intensity is fitted mapping, and it is as follows to obtain correlativity formula:
Y=-17517*exp (- x/1168) -2222*exp (- x/40)+20450
R2=0.97331
Wherein, x be hydrogen peroxide concentration, unit be μM, y is relative luminous intensity.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20060116418A (en) * | 2005-05-10 | 2006-11-15 | 고려대학교 산학협력단 | Genetic construct for detection oxidative stress and biosensor using the same |
| CN105866206A (en) * | 2016-04-13 | 2016-08-17 | 国家纳米科学中心 | Method for in-situ and real-time detection of concentration of hydrogen peroxide in living cells |
| CN109211883A (en) * | 2017-07-03 | 2019-01-15 | 苏州长光华医生物医学工程有限公司 | The chemiluminometry of concentration of hydrogen peroxide in a kind of detection solution |
| CN110044861A (en) * | 2019-05-09 | 2019-07-23 | 南宁师范大学 | The detection method of concentration of hydrogen peroxide |
-
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- 2019-08-07 CN CN201910725741.9A patent/CN110423773B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20060116418A (en) * | 2005-05-10 | 2006-11-15 | 고려대학교 산학협력단 | Genetic construct for detection oxidative stress and biosensor using the same |
| CN105866206A (en) * | 2016-04-13 | 2016-08-17 | 国家纳米科学中心 | Method for in-situ and real-time detection of concentration of hydrogen peroxide in living cells |
| CN109211883A (en) * | 2017-07-03 | 2019-01-15 | 苏州长光华医生物医学工程有限公司 | The chemiluminometry of concentration of hydrogen peroxide in a kind of detection solution |
| CN110044861A (en) * | 2019-05-09 | 2019-07-23 | 南宁师范大学 | The detection method of concentration of hydrogen peroxide |
Non-Patent Citations (3)
| Title |
|---|
| GAO ET AL.: "Constitutive Expression of a Nag-like Dioxygenase Gene through an Interal Promoter in the 2-Chloronitrobenzene Catabolism Gene Cluster of Pseudomonas stutzeri ZWLR2-1", 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》 * |
| KRAYL ET AL.: "Use of proteomics and physiological characteristics to elucidate ecotoxic effects of methyl tert-butyl ether in Pseudomonas putida KT2440", 《PROTEOMICS》 * |
| ZHANG ET AL.: "Transcriptional activation of multiple operons involved in para-nitrophenol degradation by Pseudomonas sp. Strain WBC-3", 《APPLIED AND ENVIRONMEMNTAL MICROBIOLOGY》 * |
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